cAMP compartmentalization provides a new conceptual framework to generate signaling specificity and transduction efficiency. The mechanisms involved in its establishment, maintenance and how cAMP effectors are targeted to it are not completely understood. Using thyroid cells we identified a new sub-membrane compartment, where the ERM protein radixin scaffolds both cAMP effectors Epac and PKA into a ternary complex. Maneuvers that disrupt this compartmentalization abrogate TSH/cAMP-mediated proliferation, and served as the basis for the development of new pathway-specific inhibitors. Interestingly, expression of constitutively active Rap1 but only in its phosphorylated form (G12V-S179D) rescues this inhibition, indicating the main role of this compartment is to position cAMP effectors in a local area of high cAMP concentration (i.e. a microdomain) to maximize effector activation. A sequential order of Epac-mediated activation followed by PKA-mediated phosphorylation was demonstrated. This sets an allosteric switch where pRap1 dissociates from its GEF promoting its association with new phospho-dependent binding partners, i.e. CAP1 (Cyclase- Associated Protein 1). Our preliminary studies are consistent with pRap1-CAP1 positively modulating the localized rate of cAMP synthesis. We advance here the hypothesis of a positive feedback loop as the mechanistic basis assuring that local cAMP levels in the microdomain are optimized for efficient effector activation. Defining the mechanisms involved in the pS179-dependent allosteric switch is therefore critical for the understanding of the phospho-dependent Rap1-CAP control of cAMP dynamics. We will accomplish this in two integrated aims. In Aim #1 NMR approaches will be utilized to address the mechanism involved in the phospho-dependent allosteric communication aiming at the identification of the residues that are allosterically coupled, the population of the states and their exchange dynamics. In Aim #2 a combination of biochemical and live cell imaging techniques will be used to characterize the phospho-dependent Rap1-CAP1 interaction and its ability to positively modulate compartmentalized cAMP synthesis. The long-term goal of this proposal is to understand the spatial and temporal regulation of the cAMP-dependent signaling events, and the role of Rap1 and its phosphorylation state as a signal integration unit. Understanding the mechanisms responsible for cAMP compartmentalization will eventually provide insights into the rational design of new specific inhibitors with effector pathway selectivity.